S Alex Mitsialis, PhD | Harvard Medical School (original) (raw)

Papers by S Alex Mitsialis, PhD

Circulation, Nov 27, 2012

Background-Hypoxia induces an inflammatory response in the lung manifested by alternative activat... more Background-Hypoxia induces an inflammatory response in the lung manifested by alternative activation of macrophages with elevation of proinflammatory mediators that are critical for the later development of hypoxic pulmonary hypertension. Mesenchymal stromal cell transplantation inhibits lung inflammation, vascular remodeling, and right heart failure and reverses hypoxic pulmonary hypertension in experimental models of disease. In this study, we aimed to investigate the paracrine mechanisms by which mesenchymal stromal cells are protective in hypoxic pulmonary hypertension. Methods and Results-We fractionated mouse mesenchymal stromal cell-conditioned media to identify the biologically active component affecting in vivo hypoxic signaling and determined that exosomes, secreted membrane microvesicles, suppressed the hypoxic pulmonary influx of macrophages and the induction of proinflammatory and proproliferative mediators, including monocyte chemoattractant protein-1 and hypoxia-inducible mitogenic factor, in the murine model of hypoxic pulmonary hypertension. Intravenous delivery of mesenchymal stromal cell-derived exosomes (MEX) inhibited vascular remodeling and hypoxic pulmonary hypertension, whereas MEX-depleted media or fibroblast-derived exosomes had no effect. MEX suppressed the hypoxic activation of signal transducer and activator of transcription 3 (STAT3) and the upregulation of the miR-17 superfamily of microRNA clusters, whereas it increased lung levels of miR-204, a key microRNA, the expression of which is decreased in human pulmonary hypertension. MEX produced by human umbilical cord mesenchymal stromal cells inhibited STAT3 signaling in isolated human pulmonary artery endothelial cells, demonstrating a direct effect of MEX on hypoxic vascular cells. Conclusion-This study indicates that MEX exert a pleiotropic protective effect on the lung and inhibit pulmonary hypertension through suppression of hyperproliferative pathways, including STAT3-mediated signaling induced by hypoxia.

International Journal of Molecular Sciences, Aug 27, 2018

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the... more Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of the pulmonary arteries, increased pulmonary infiltrates, loss of vascular cross-sectional area, and elevated pulmonary vascular resistance. Despite recent advances in the management of PAH, there is a pressing need for the development of new tools to effectively treat and reduce the risk of further complications. Dysregulated immunity underlies the development of PAH, and macrophages orchestrate both the initiation and resolution of pulmonary inflammation, thus, manipulation of lung macrophage function represents an attractive target for emerging immunomodulatory therapies, including cell-based approaches. Indeed, mesenchymal stem cell (MSC)-based therapies have shown promise, effectively modulating the macrophage fulcrum to favor an anti-inflammatory, pro-resolving phenotype, which is associated with both histological and functional benefits in preclinical models of pulmonary hypertension (PH). The complex interplay between immune system homeostasis and MSCs remains incompletely understood. Here, we highlight the importance of macrophage function in models of PH and summarize the development of MSC-based therapies, focusing on the significance of MSC exosomes (MEx) and the immunomodulatory and homeostatic mechanisms by which such therapies may afford their beneficial effects.

Springer eBooks, 2002

Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes p... more Acute hypoxia in the lung causes arteriolar vasoconstriction whereas prolonged hypoxia promotes proliferation and migration of vascular smooth muscle cells (VSMC) and extracellular matrix deposition in the arterial wall, a process known as vascular remodeling.1 These abnormalities are characteristic of pulmonary hypertension.2 Several clinical conditions characterized by lung inflammation have been linked to the development of chronic pulmonary hypertension.3 Interestingly, perivascular inflammatory cell infiltration as well as increased serum levels of pro-inflammatory cytokines, such as interleukin (IL)-lβ and IL-6, have been reported in clinical cases of primary pulmonary hypertension.4-5 However, little attention has been given up to now to the role of pulmonary inflammation in the pathogenesis of pulmonary hypertension induced by hypoxia.

Journal of Immunology, May 1, 2020

Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macro... more Supplemental Figure 1. Morphological and phenotypic characterization of bone marrow-derived macrophages (BMDM) from 6-8 week old FVB mice by flow cytometry. Supplemental Figure 2. Detection of P50 nuclear staining (white arrows) in alveolar macrophages obtained from Hmox1+/+ and Hmox1-/- mice at baseline normoxic conditions and expression of cytokines and markers of alveolar macrophage activation in normoxic Hmox1+/+ and Hmox1-/- mice as determined by RT-qPCR.

Journal of Virology, 1982

Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cl... more Avian tumor virus supercoiled DNA was isolated from infected quail tumor cells and molecularly cloned in pBR322. Four different recombinant clones denoted pATV-6, pATV-7, pATV-8, and pATV-9 were characterized in detail by restriction endonuclease mapping and by DNA sequencing. The results of these studies indicate that (i) the two large terminal repeats (LTRs) present in PATV-6, are different sizes, (ii) pATV-8 and pATV-9 contain only one LTR, (iii) pATV-7 contains an inversion of 0.6 kilobase in the env gene and a deletion of the U3 region and the src gene, and (iv) the src gene is deleted in pATV-6 and pATV-9. Circle formation from linear molecules was also examined in several of the clones by DNA sequencing through the circle joint. pATV-6 is an example of one class of circular molecules and contains a partially repeated LTR similar to that reported by Ju and Skalka (Cell 22:379-386, 1980). A second class of circles was exemplified by pATV-8 and pATV-9, which contain a single cop...

Journal of Virology, 1980

For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates t... more For the first time, we present evidence with restriction enzymes HpaII and MspI which indicates that the proviral DNA sequence of avian sarcoma virus is modified by methylation in a nonpermissive rat cell line but not in permissive chicken cells. Some of the endogenous viral sequences in the permissive cells were also methylated. No 5-methylcytosine could be detected in the unintegrated viral DNA.

Experimental Biology and Medicine, 2003

An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammati... more An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and bilirubin, downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment or by repressing the induction of cytokines and chemokines. In the present study we used genetically engineered mice that express high levels of a human cDNA HO-1 transgene in lung epithelium to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) challenge. We found that both mRNA and protein levels of specific cytokines and chemokines were significantly elevated in response to hypoxia in the lungs of wild-type mice after 2 and 5 days of exposure but significantly suppressed in the hypoxic lungs of transgenic mice, su...

The FASEB Journal, 2004

We have previously reported that hypoxia induces a pronounced inflammatory response in the mouse ... more We have previously reported that hypoxia induces a pronounced inflammatory response in the mouse lung associated with elevated levels of specific chemokines. To further explore the mechanisms involved in lung inflammation, we exposed RAW 264.7 cells as well as mouse primary macrophages to hypoxia and analyzed chemokine gene expression. Among the genes examined, macrophage inflammatory protein-2 (MIP-2) expression was prominently induced by hypoxia both at the mRNA and the protein level. When RAW 264.7 cells were transfected with a panel of plasmids harboring a luciferase marker gene under the control of wild-type or mutant variants of the MIP-2 gene promoter, a strong hypoxic induction of expression (9-to 17-fold) was observed. This induction was abolished by a mutation targeted to an NF-κB binding site in the MIP-2 promoter. Concordantly, specific NF-κB binding to the cognate sequence was enriched in nuclear extracts from hypoxic but not normoxic RAW 264.7 cells. The mechanism of MIP-2 gene induction by hypoxia was further characterized using inhibitors of signaling kinases. Inhibition of the p42/p44 and PI3 kinases but not p38 MAPK abolished the NF-κB-driven upregulation of MIP-2 gene expression by hypoxia. This attenuation of the NF-κB response to hypoxia did not involve decreased nuclear NF-κB abundance but correlated with diminished transactivation potential of the p65 subunit. Our results indicate that the hypoxic signal for induction of MIP-2 gene expression is implemented through enhanced NF-κB activity and transmitted along the p42/44 and PI3 kinase pathways. Key words: chemokines • signaling pathways ypoxia is a critical stimulus of several pathophysiological conditions including erythropoiesis, angiogenesis, and pulmonary vascular remodeling (1, 2). We have recently reported that exposing mice to prolonged hypoxia (2-5 days) results in marked pulmonary inflammation. This inflammatory response is characterized by enhanced leukocyte infiltration into the alveolar space and extremely high levels of cytokines and chemokines in the hypoxic lung (3). H

Proceedings of the National Academy of Sciences, 1992

The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the ... more The Drosophila chorion factor 1/ultraspiracle (CF1/USP) transcription factor, a homologue of the retinoid X receptor, is a developmentally important member of the family of nuclear (steroid) hormone receptors. Using newly developed monoclonal antibodies and a full-length bacterially produced protein, we have studied in detail the in vitro DNA-binding properties of this factor and aspects of its distribution in vivo. During oogenesis, CF1/USP is present both in germline cells and in the somatic follicular epithelium. We have determined the optimal binding site of partially purified bacterially produced CF1/USP by an in vitro selection procedure and also have characterized its binding to the follicular-specific chorion s15 promoter. In vitro this bacterially produced factor is unusual in binding to a single element ("half-site"); simultaneous but noncoordinate binding to a second half-site is possible if these repeated elements are organized in direct orientation and spaced ...

American Journal of Respiratory Cell and Molecular Biology, 1995

Retinoids have been shown to influence pattern formation during development and regeneration in n... more Retinoids have been shown to influence pattern formation during development and regeneration in numerous systems such as limbs, vertebrae, and neural tube although there is little information about the effects of retinoids on pattern formation in visceral organs. We investigated the effects of exogenous retinoic acid on the in vitro pattern of airway branching and on lung epithelial cell differentiation. Histology, [3H]thymidine autoradiographies and reverse transcriptase/polymerase chain reaction (RT/PCR) amplification were used to assess the effects of retinoids and the expression of lung epithelial markers of differentiation. We found that retinoic acid interferes, in a dose-dependent fashion, with the expression of epithelial genes that are found in distal segments of the fetal lung (surfactant-associated proteins SP-A, SP-B, and SP-C). At high concentrations, retinoic acid (RA) dramatically altered the developmental pattern of the lung, favoring growth of structures that resemble proximal airways and concomitantly suppressing distal epithelial buds. We hypothesize that this in vitro "proximalizing" effect on the developing lung may be related to alterations in the expression of pattern-related genes.

Fredenburgh et al. – 1 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, is a cytop... more Fredenburgh et al. – 1 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, is a cytoprotective enzyme that plays a central role in the defense against oxidative and inflammatory insults in the lung. HO-1 catalyzes the degradation of heme, a potent oxidant, into biliverdin, iron, and carbon monoxide (CO). These downstream products of heme catabolism have recently been found to mediate the anti-oxidant, anti-apoptotic, anti-proliferative, vasodilatory and antiinflammatory properties of HO-1. While absence of HO-1 is rare in humans, a number of HO-1 promoter polymorphisms have been identified which may influence HO-1 expression in vivo and lead to disease states. This review will summarize studies that implicate HO-1 and heme metabolites in the pathophysiology of pulmonary disease and discuss recent advances in the therapeutic applications of HO-1.The Role of HO-1 in Pulmonary Disease Fredenburgh et al. – 2 Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, pla...

American Journal of Physiology-Lung Cellular and Molecular Physiology, 2019

Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be m... more Exposure to hypoxia causes an inflammatory reaction in the mouse lung, and this response can be modulated by overexpressing the hypoxia-inducible stress-response enzyme, heme oxygenase-1 (HO-1). We hypothesized that the inflammasome activity may be a central pathway by which HO-1 controls pulmonary inflammation following alveolar hypoxia. Therefore, we investigated whether HO-1 controls inflammasome activation by altering its expression in macrophages primed with classic NOD-like receptor containing a pyrin domain 3 (NLRP3) inducers, and in murine lungs lacking HO-1 and exposed to acute hypoxia. We found that lack of HO-1 activated lipopolysaccharide (LPS) and ATP-treated bone marrow-derived macrophages, causing an increase in secreted levels of cleaved interleukin (IL)-1B, IL-18, and caspase-1, markers of increased inflammasome activity, whereas HO-1 overexpression suppressed IL-1B, NLRP3, and IL-18. The production of cleaved IL-1B and the activation of caspase-1 in LPS- and ATP-pr...

Idiopathic interstitial pneumonias, 2018

Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models o... more Rationale: Mesenchymal stromal/stem cell (MSC) therapy has shown promise in experimental models of idiopathic pulmonary fibrosis (IPF), a chronic progressive lung disease characterized by interstitial fibrosis with decreasing lung volumes and hypoxemic respiratory failure. Recently, we reported that the therapeutic capacity of MSCs predominantly resides in the secretome, and that the chief therapeutic vector therein is represented by the exosomes. Objectives: To test the therapeutic effects of MSC-exosomes (MEx) in a bleomycin-induced pulmonary fibrosis model and investigate putative mechanisms of action. Methods: Exosomes were isolated from media conditioned by human bone marrow MSCs (MEx). Adult mice (C57BL/6 strain) were challenged with endotracheal instillation of bleomycin and treated with MEx concurrently or at day 7. Treated animals and appropriate control groups were assessed at day 7 and/or day 14. Results: Bleomycin-challenged mice presented with severe septal thickening and prominent fibrosis, and this was effectively prevented (day 0 treatment) or reversed (day 7 treatment) by a single dose of MEx. Furthermore, MEx therapy modulated whole lung macrophage phenotype, and shifted the proportion of lung ‘proinflammatory’ classical monocytes, ‘regulatory’ monocytes and alveolar macrophages to favor the monocyte/macrophage profiles of untreated-control mice, and, importantly a parallel immunomodulatory effect was demonstrated in the bone marrow. Notably, transplantation of MEx-preconditioned bone marrow-derived monocytes alleviated core features of pulmonary fibrosis. Conclusion: A bolus dose of MEx prevents and reverts core features of bleomycin-induced pulmonary fibrosis. The beneficial actions of MEx are mediated via the systemic modulation of monocyte phenotypes.

Cellular & molecular biology research, 1995

Members of the E2F gene family are transcription factors that have been implicated in the control... more Members of the E2F gene family are transcription factors that have been implicated in the control of genes essential for cell cycle progression. Regulation of E2F function is finely tuned by the retinoblastoma tumor suppressor gene product and a small family of related "pocket proteins," with the participation of a number of cyclins and cyclin-dependent kinases. Perturbations of this regulatory network can lead to oncogenic transformation and, in certain systems, to the loss of the ability to maintain terminal differentiation. We describe here the cloning, structural characterization, and tissue expression pattern of a new member of the E2F family, E2F-5. We show that this protein is highly conserved between human and rat but exhibits considerable divergence from E2F-1, E2F-2, or E2F3. Together with the recently reported E2F-4, E2F-5 defines a new branch of the E2F family. The distribution of E2F-5 mRNA among adult rat tissues and the temporal pattern of its expression dur...

Journal of General Virology, 1983

The structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC ... more The structure and arrangement of the multiple provirus copies of avian sarcoma virus in a rat XC cell line were studied by restriction endonucleases. The following observations were made: (i) the majority of the proviruses integrated randomly with respect to cell DNA; (ii) no gross deletions or rearrangements in the proviruses were observed; (iii) two types of proviruses (type I and type II) could be distinguished on the basis of restriction endonuclease cleavage sites; (iv) the virus rescued from these cells was derived from type II provirus, which has a novel EcoRI site between the env andpol genes; (v) most of the provirus units contained the src gene.

Additional file 4. TSC possess the capability of clone formation and differentiation of into alve... more Additional file 4. TSC possess the capability of clone formation and differentiation of into alveolar epithelial cells in vitro. TSCs were cultured in a 100-mm dish at limited dilution of one cell every 60mm2 for 14 days. Representative phase contrast images of an entire clone (a), and a clone immunofluorescence stained for CD117 (green) and DAPI (blue, b). TSCs were seeded on the apical side of a clear12-transwell plate and exposed to differentiation medium of 2% FBS DMEM/F12 supplied with 0.005mg/mL insulin, 0.01mg/mL Tranferrin, 30nM Sodium selenite, 10nM Hydrocortisone, 10nM Beta-estradiol, 10nM HEPES, 2mM L-glutamine and 50ng/mL EGF for 6 days, following air-liquid interface for an additional 11-12 days. Representative images of these differentiated cells immuno-fluorescence stained for AQP5 (red, c and d), SPC (red, e and f). Scale bars represent 1000µm for a and b, 50µm for c~f. Bar graphs showing quantitation of qRT-PCR for AQP5 (g) and SPC (h) in fold change comparing diffe...